The recurrent outbreak of deadly infectious diseases worldwide heightens the urgency to develop new and improved vaccines to combat their spread. Unfortunately, many of the current vaccines, or those in development, induce suboptimal immune responses and require large antigen doses, multiple immunizations, or periodic boosting to provide long-term protection. It is known that adjuvant combinations can increase the efficacy and safety of vaccines, promote dose sparing, and, possibly, contribute to more rapid and durable immune protection against pathogens?in particular those that target multiple receptors and pathways. Studies to date, however, have focused on individual adjuvants; needed now is a deeper understanding of the mechanisms by which combination adjuvants produce their effects. At this time, aluminum-containing adjuvants (alum) are the most popular because of their strong and long-lasting immunostimulatory effects and long-standing safety record; they are also low-cost. But these adjuvants promote primarily Th2-biased humoral immune responses, not the strong Th1 and/or CD8+ cell-mediated immune responses needed for vaccines targeting intracellular pathogens. To meet this need, we propose to test a new adjuvant combination?a novel protein adjuvant (ASP-1) and the traditional adjuvant alum?and study the mechanisms by which it induces its effects when formulated with the receptor-binding domain (RBD) in the spike (S) protein of Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV), a viral pathogen with a high mortality rate and high pandemic potential. The ASP-1 adjuvant, a naturally occurring secreted protein of Onchocerca volvulus shown to have intrinsic immunostimulatory properties on innate cells, can promote a balanced antibody response and Th1-biased cellular responses to several soluble vaccine candidate antigens, as well as commercial inactivated viral vaccines. It also promotes dose sparing when combined with a commercial trivalent influenza vaccine, and confers protection in young and aged adult mice after one immunization against homologous or heterologous influenza virus challenge. As important, antibodies produced to it during vaccinations do not limit its utility or adjuvanticity n any setting tested. We will study the effect of the physical association between ASP- 1 and alum on their adjuvanticity (adsorption to alum is possibly essential here) by comparing the immunogenicity in vivo when formulated with MERS-RBD. Upon identification of the most effective ASP- 1/alum/RBD formulation, we will determine the mechanism of action of this novel adjuvant combination, both in vitro and in vivo, to determine the precise interactions among the various innate and adaptive cell types, chemokines, and cytokines that they stimulate in the injection site and in the draining lymph nodes responsible for potentiating the enhanced immunogenicity elicited by the ASP-1 and alum-adjuvanted MERS-RBD vaccine. Looking forward, we hope to develop a highly effective adjuvant combination safe for use in humans, one that will boost distinctive immune responses against various pathogens with a minimal amount of the vaccine immunogen.